Secreted protein therapeutics and uses thereof

ABSTRACT

A targeting fusion protein comprising a component that comprises a (i) ligand or derivative or fragment thereof that binds a pre-selected target surface protein, such as a receptor, and (ii) an active agent or therapeutic agent(s), and further optionally (iii) a multimerizing component and/or (iv) a signal sequence. In a preferred embodiment, the targeting fusion polypeptide targets muscle and is useful to treat a muscle-related disease or condition, such as muscle atrophy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC § 119(e) of U.S.Provisionals 60/507,168 filed 30 Sep. 2003, 60/516,806 filed 3 Nov.2003, 60/573,525 filed 21 May 2004, 60/534,819 filed 7 Jan. 2004,60/584,956 filed 2 Jul. 2004, 60/529,826 filed 16 May 2003, 60/534,654filed 7 Jan. 2004, 60/581,833 filed 22 Jun. 2004, and 60/554,640 filed19 Mar. 2004, which applications are herein specifically incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to therapeutic fusion proteins, methods ofproducing such fusion proteins, and methods for treating, diagnosing, ormonitoring diseases or conditions using these proteins.

BRIEF SUMMARY OF THE INVENTION

In the broadest embodiment, the present invention comprises compositionsand methods for delivering one or more active or therapeutic agent(s).In one embodiment, the present invention provides compositions andmethods for specifically delivering one or more active or therapeuticagent(s) to a pre-selected target site. In this embodiment, the presentinvention provides fusion polypeptides capable of delivering one or moreactive or therapeutic agent(s) to a target site defined by the presenceof one or more expressed cell surface proteins. The targeting fusionpolypeptides are therapeutically useful, as well as useful in a varietyof in vitro and in vivo diagnostic and prognostic assays.

In a first aspect, the invention provides a targeting fusion polypeptidecomprising (i) a targeting ligand, or derivative or fragment thereof,capable of binding specifically to a pre-selected cell surface protein,and (ii) an active or therapeutic agent. In specific embodiments, thetargeting fusion polypeptide optionally further comprises (iii) amultimerizing component capable of forming a multimer with anothertargeting fusion polypeptide, and/or (iv) a signal sequence.

In a second aspect, the invention provides a muscle-targeting fusionpolypeptide, comprising (i) a targeting ligand, or derivative orfragment thereof, capable of binding specifically to a muscle cellsurface protein, and (ii) an active or therapeutic agent. In specificembodiments, the muscle-targeting fusion polypeptide optionally furthercomprises (iii) a multimerizing component capable of forming a multimerwith another targeting fusion polypeptide, and/or (iv) a signalsequence.

In specific embodiments, the muscle-targeting ligand specifically bindsa muscle surface protein, such as a receptor. In a more specificembodiment, the muscle surface receptor is MuSK. In an even morespecific embodiment, the muscle-targeting fusion polypeptidespecifically targets skeletal muscle, and comprises a MuSK ligand, orfragment of a MuSK ligand capable of binding the MuSK receptor. Inspecific embodiments, the MuSK-specific ligand is agrin or a fragment orderivative thereof capable of binding MuSK, or an anti-MuSK antibody orfragment or derivative thereof, including, for example, an scFv.

In other specific embodiments, the muscle-targeting ligand of themuscle-targeting fusion polypeptide comprises three or more musclecadherin (M-cadherin) extracellular cadherin domains, or derivatives orfragments thereof, capable of binding specifically to a muscle cells orother cells that express homophilic muscle cadherins. In one specificembodiment, the muscle-targeting ligand consists essentially of thefirst three (3) or four (4) N-terminal extracellular domains ofM-cadherin.

The active or therapeutic agent may be any agent that is desirable todeliver to a pre-selected site for therapeutic purposes. In specificembodiments, the active or therapeutic agent is a ligand for a secondcell surface receptor, and is capable of binding and activating a secondreceptor. In other embodiments, the active or therapeutic agent is anagent capable of blocking the activity of another agent that is activeon the target cell. In a specific embodiment, the active or therapeuticagent is selected from the group consisting of IL-15, myotrophin,urocortin, urocortin 11, a natural or mutant IGF-1 or IGF-2, insulin,the pro domain of myostatin, hGH, proliferin, follistatin, FSTL1, andFLRG, and a biologically active fragments thereof.

In one specific embodiment, the invention provides a muscle-targetingfusion polypeptide, comprising (i) agrin, or a fragment or derivativethereof capable of binding the MuSK receptor; and (ii) an active ortherapeutic agents selected from the group consisting of IL-15,myotrophin, urocortin, urocortin II, a natural or mutant IGF-1 or IGF-2,insulin, the pro domain of myostatin, hGH, proliferin, follistatin,FSTL1, and FLRG, or a biologically active fragment thereof; andoptionally (iii) a multimerizing component, and/or (iv) a signalsequence. In another specific embodiment, the invention provides amuscle-targeting fusion polypeptide comprising (i) three (3) N-terminalextracellular domains of M cadherin; and (ii) an active or therapeuticagents selected from the group consisting of IL-15, myotrophin,urocortin, urocortin II, a natural or mutant IGF-1 or IGF-2, insulin,the pro domain of myostatin, hGH, proliferin, follistatin, FSTL1, andFLRG, or a biologically active fragment thereof; and optionally (iii) amultimerizing component, and/or (iv) a signal sequence.

In a separate embodiment, the invention provides composition fordelivering two active or therapeutic agents. The two active ortherapeutic agents may act synergistically when present together.Accordingly, in a third aspect, the invention features a fusionpolypeptide comprising (i) a first active or therapeutic agent, and (ii)a second active or therapeutic agent. In specific embodiments, thefusion polypeptide optionally further comprises (iii) a multimerizingcomponent capable of forming a multimer with another targeting fusionpolypeptide, and/or (iv) a signal sequence. In a preferred embodiment,one active or therapeutic agent is natural or mutant human growthhormone (hGH) or proliferin, or a biologically active fragment thereofand the other agent is natural or mutant IGF-1 or IGF-2, or abiologically active fragment thereof.

In another embodiment, the invention provides compositions fordelivering IGF-related polypeptides, which may be used alone or as acomponents of the targeting or therapeutic fusion molecules describedherein. Such polypeptides comprise a mutant IGF-1 or IGF-2 moleculehaving (i) deletion of the first three amino acids (Δ3), or substitutionof either arginine (R) or alanine (A) for glutamic acid (E) at position3 (E3R or E3A); and (ii) modification in the double arginines (RR) atpositions 36 and 37 by either substitution of an alanine (A) for anarginine (A) at position 36 or 37 or deletion of R36(del36) orR37(del37); wherein the IGF-derived polypeptides optionally comprise(iii) a multimerizing component capable of forming a multimer withanother IGF-related polypeptide, and/or (iv) a signal sequence.

In other embodiment, the invention provides compositions for deliveringmyostatin-inhibiting fusion polypeptides, which may be used alone or ascomponents of the targeting or therapeutic fusion molecules describedherein. Such compositions comprise the propeptide of human myostatin. Inone specific embodiment, the fusion proteins comprise the propeptide ofmyostatin fused to a multimerizing component, such as the Fc domain ofIgG.

In a fourth aspect, the invention features a targeting fusionpolypeptide comprising (i) a targeting ligand, or derivative or fragmentthereof, capable of binding specifically to a pre-selected cell surfaceprotein, (ii) a first active or therapeutic agent, and (ii) a secondactive or therapeutic agent. In specific embodiments, the fusionpolypeptide optionally further comprises (iii) a multimerizing componentcapable of forming a multimer with another targeting fusion polypeptide,and/or (iv) a signal sequence. In a preferred embodiment, one active ortherapeutic agent is natural or mutant human growth hormone (hGH) orproliferin, or a biologically active fragment thereof and the otheragent is natural or mutant IGF-1 or IGF-2, or a biologically activefragment thereof. In one preferred embodiment, the targeting ligand isagrin, one active agent is IGF1 or mutant thereof, and one active agentis hGH, such as in the targeting fusion polypeptides of SEQ IDNOS:23-26.

In one embodiment, the targeting fusion polypeptide is amuscle-targeting fusion polypeptide, comprising (i) a targeting ligand,or derivative or fragment thereof, capable of binding specifically to amuscle cell surface protein, (ii) a first active or therapeutic agent,and (ii) a second active or therapeutic agent, optionally furthercomprises (iii) a multimerizing component capable of forming a multimerwith another targeting fusion polypeptide, and/or (iv) a signalsequence. In a preferred embodiment, one active or therapeutic agent isnatural or mutant human growth hormone (hGH), or a biologically activefragment thereof and another active or therapeutic agent is natural ormutant IGF-1 or IGF-2, or a biologically active fragment thereof.

In a specific embodiment, the muscle-targeting fusion polypeptidecomprises (i) agrin, or a fragment or derivative thereof capable ofbinding the MuSK receptor; and (ii) a first active or therapeutic agent,wherein the first active or therapeutic agent is natural or mutant humangrowth hormone or a biologically active fragment thereof; (iii) a secondactive or therapeutic agent, wherein the second active or therapeuticagent is natural or mutant IGF-1 or IGF-2, or a biologically activefragment thereof; and optionally (iv) a multimerizing component, and/or(v) a signal sequence. In another embodiment, the muscle-targetingfusion polypeptide comprises a sequence selected from the groupconsisting of SEQ ID NOS: 9-31. In preferred embodiments, themuscle-targeting fusion polypeptide comprises a sequence selected fromthe group consisting of SEQ ID NOS:26-29.

In a fifth aspect, the invention provides an IGF-related polypeptidecomprising a mutant IGF-1 or IGF-2 molecule having (i) deletion of thefirst three amino acids (Δ3), or substitution of either arginine (R) oralanine (A) for glutamic acid (E) at position 3; and (ii) modificationin the double arginines (RR) at positions 36 and 37 by eithersubstitution of an alanine (A) at position 36 or position 37 or deletionor R36 or R37; wherein the IGF-derived polypeptides optionally comprise(iii) a multimerizing component capable of forming a multimer withanother IGF-related polypeptide, and/or (iv) a signal sequence.

In a sixth aspect, the invention provides a fusion polypeptidecomprising the propeptide of human myostatin fused to a multimerizingcomponent, such as the Fc domain of IgG.

In specific embodiments wherein the fusion polypeptides of the inventioncomprise a multimerizing component, the multimerizing component may beselected from the group consisting of (i) a multimerizing componentcomprising a cleavable region (C-region), (ii) a truncated multimerizingcomponent, (iii) an amino acid sequence between 1 to about 500 aminoacids in length, optionally comprising at least one cysteine residue,(iv) a leucine zipper, (v) a helix loop motif, (vi) a coil-coil motif,(vii) an Fc-protein, and (viii) a combination thereof. In specificembodiments in which the targeting fusion polypeptide comprises a signalsequence, the signal sequence may comprise any sequence known to askilled artisan for directing secretion of a polypeptide or protein froma cell, include natural or synthetic sequences. Generally, a signalsequence is placed at the beginning or amino-terminus of the fusionpolypeptide of the invention. Such a signal sequence may be native tothe cell, recombinant, or synthetic.

The components of the fusion polypeptides of the invention may beconnected directly to each other or connected via one or more spacersequences. In one preferred embodiment, the components are fuseddirectly to each other. In another preferred embodiment, the componentsare connected with a nucleic acid sequence encoding a spacer of 1-200amino acids. Any spacer known to the art may be used to connect thepolypeptide components. A spacer sequence may also include a sequenceused to enhance expression of the fusion polypeptide, providerestriction sites, and allow component domains to form optimal tertiaryand quaternary structures and/or to enhance the interaction of acomponent with its receptor. In one embodiment, the fusion polypeptideof the invention comprises one or more peptide sequences between one ormore components which is (are) between 1-25 amino acids.

The components of the fusion polypeptide of the invention may bearranged in a variety of configurations. For example, the targetingligand component (1), the active or therapeutic agent(s) component (2),and the optional multimerizing component (3) may be arranged in one ofthe following configurations: 1-2; 2-1; 1-2-3; 1-3-2; 3-1-2; 2-1-3;2-3-1, or 3-2-1. Still further, multiple components of a targetingligand or active agent may be included in the fusion polypeptide, e.g.,1-1-2,2-1-1,1-2-1,2-2-1,1-1-2-1-1, etc.

When the active or therapeutic agent(s) component of the fusionpolypeptide of the invention comprises two active or therapeutic agents,these agents may be arranged in a variety of configurations. Forexample, active agent (1) and agent (2) may be arrange either 1-2 or2-1. Still further, the same agent may be included more than once in thecomponent, e.g., 1-1-2; 1-2-2; 1-2-1, etc.

In a seventh aspect, the invention provides nucleic acid moleculesencoding the fusion polypeptides of the invention.

In related aspects, the invention features a vector comprising a nucleicacid molecule of the invention, including expression vectors comprisingthe nucleic acid molecules operatively linked to an expression controlsequence, and host-vector systems for the production of a fusionpolypeptide which comprise the expression vector, in a suitable hostcell; host-vector systems wherein the suitable host cell is, withoutlimitation, a bacterial, yeast, insect, or mammalian cell. Examples ofsuitable cells include E. coli, B. subtilis, BHK, COS and CHO cells.Additionally encompassed are fusion polypeptides of the inventionmodified by acetylation or pegylation. Methods for acetylating orpegylating a protein are well known in the art.

The invention further provides a method of producing a fusionpolypeptide of the invention, comprising culturing a host celltransfected with a vector comprising a nucleic acid molecule of theinvention, under conditions suitable for expression of the protein fromthe host cell, and recovering the polypeptide so produced.

The invention features therapeutic methods for the treatment of adisease or condition, comprising administering a therapeuticallyeffective amount of a fusion protein of the invention to a subject inneed thereof, or a subject at risk for development of that disease orcondition. When the disease or condition is a muscle condition, such asatrophy, the therapeutic method of the invention comprises administeringa therapeutically effective amount of a muscle-targeting fusion proteinof the invention to a subject in need thereof, wherein themuscle-related disease or condition is ameliorated or inhibited. In onespecific embodiment, the invention features a method of inhibiting orameliorating muscle atrophy, comprising administering a therapeuticallyeffective amount of a fusion polypeptide comprising IGF-1 and agrin, orbiologically active fragments or derivatives of IGF-1 and/or agrin,wherein IGF-1 is specifically delivered to the desired site by agrinbinding to the muscle-specific surface receptor MuSK. In anotherembodiment, the muscle-specific fusion polypeptide comprises agrin andIL-15, or biologically active fragments or derivatives of agrin and/orIL-15. In another embodiment, the muscle-specific fusion polypeptidecomprises agrin; natural or mutant hGH, or a biologically activefragment or a derivative thereof; and natural or mutant IGF-1, or abiologically active fragment or a derivative thereof. In yet anotherembodiment, the muscle-specific fusion polypeptide comprises agrin;natural or mutant hGH, or a biologically active fragment or a derivativethereof; and natural or mutant IGF-2, or a biologically active fragmentor a derivative thereof. The muscle-related condition or disordertreated by the fusion polypeptides of the invention may arise from anumber of sources, including for example: denervation; degenerative,metabolic or inflammatory neuropathy; infantile and juvenile spinalmuscular atrophies; autoimmune motor neuropathy; from chronic disease,including cachexia resulting from cancer, AIDS, fasting orrhabdomyolysis; and from muscular dystrophy syndromes such as Duchenne.

Accordingly, in a seventeenth aspect, the invention featurespharmaceutical compositions comprising a targeting fusion protein of theinvention with a pharmaceutically acceptable carrier. Suchpharmaceutical compositions may comprise the fusion proteins or nucleicacids which encode them. In specific embodiments, the pharmaceuticalcomposition of the invention comprises a muscle-targeting polypeptidecomprising (i) agrin, or a biologically active fragment or derivativethereof, capable of binding the muscle-specific surface protein MuSK,and (ii) an active agent selected from the group consisting of insulinlike growth factor 1 (IGF-1) and interleukin-15 (IL-15), optionallyfurther comprising (iii) a multimerizing component, together with apharmaceutically acceptable carrier. In another embodiment, thepharmaceutical composition of the invention comprises a muscle-targetingpolypeptide comprising (i) agrin, or a fragment or derivative thereofcapable of binding the MuSK receptor; and (ii) a first active ortherapeutic agent, wherein the first active or therapeutic agent isnatural or mutant human growth hormone or proliferin, or a biologicallyactive fragment thereof; (iii) a second active or therapeutic agent,wherein the second active or therapeutic agent is natural or mutantIGF-1 or IGF-2, or a biologically active fragment thereof; andoptionally (iv) a multimerizing component, together with apharmaceutically acceptable carrier.

The invention features a method of activating and/or phosphorylationmultiple cell surface receptors simultaneously, by providing a fusionpolypeptide capable of binding multiple cell surface receptor to a cellexpressing multiple receptors. In one embodiment, the invention featuresa fusion polypeptide capable of binding both MuSK and IGF-R. In anotherembodiment, the invention provides a fusion polypeptide capable ofbinding both GHR and IFG-R. In yet another embodiment, the inventionprovides a fusion polypeptide capable of binding MuSK, GHR, and IGF-Rsimultaneously. Preferably, the method of the invention is achieved byproviding a targeting fusion polypeptide described above.

Other objects and advantages will become apparent from a review of theensuing detailed description.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “a method”include one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference.

General Description

The invention encompasses fusion polypeptides and nucleic acids thatencode them which comprise one or more active or therapeutic agent(s).The invention further provides targeting fusion polypeptides and nucleicacids which encode then comprise a targeting ligand that specificallybinds a pre-selected cell surface protein, such as a receptor, and oneor more active or therapeutic agent(s) capable of achieving a desiredeffect when delivered to the desired cell or tissue.

Definitions

“Biologically active” fragments or derivatives of a targeting ligand oran active or therapeutic component of the targeting fusion polypeptidesof the invention encompass any naturally occurring molecule, or mutantor derivative thereof capable of achieving the desired effect at thetarget site. For example, when the active or therapeutic agent is IGF-1,the invention envisions the use of a mutant or derivative IGF-1 moleculecapable of binding the IGF-1 receptor. Examples of such mutants, whichretain the activity of the native molecule but which have greaterstability and potency, are described herein. A “biologically active”fragment of derivative of any targeting component is any portion ormutant thereof capable of binding the target cell. Thus, for example,when the targeting ligand is agrin, a biologically active fragment orderivative is any portion or mutant of agrin capable of binding the MuSKreceptor.

The terms “treatment”, “treating”, and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease, condition, or symptoms thereof, and/ormay be therapeutic in terms of a partial or complete cure for a diseaseor condition and/or adverse effect attributable to the disease orcondition. “Treatment” as used herein covers any treatment of a diseaseor condition of a mammal, particularly a human, and includes: (a)preventing the disease or condition from occurring in a subject whichmay be predisposed to the disease or condition but has not yet beendiagnosed as having it; (b) inhibiting the disease or condition, i.e.,arresting its development; or (c) relieving the disease or condition,i.e., causing regression of the disease or condition. The population ofsubjects treated by the method of the disease includes subject sufferingfrom the undesirable condition or disease, as well as subjects at riskfor development of the condition or disease.

By the term “therapeutically effective dose” is meant a dose thatproduces the desired effect for which it is administered. The exact dosewill depend on the purpose of the treatment, and will be ascertainableby one skilled in the art using known techniques (see, for example,Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding).

As used herein, a “condition or disease” generally encompasses acondition of a mammalian host, particularly a human host, which isundesirable and/or injurious to the host. Thus, treating amuscle-related condition with a fusion polypeptide which specificallytargets skeletal muscle will encompass the treatment of a mammal, inparticular, a human, who has symptoms reflective of decreased targetmuscle receptor activation, or who is expected to have such decreasedlevels in response to a disease, condition or treatment regimen.Treating a muscle-related condition or disease encompasses the treatmentof a human subject wherein enhancing the activation of a target musclereceptor with the muscle specific fusion polypeptide of the inventionresults in amelioration of an undesirable symptom resulting from themuscle-related condition or disease. As used herein, a “muscle-relatedcondition” also includes a condition in which it is desirable to alter,either transiently, or long-term, activation of a particular targetmuscle receptor.

Targeting Fusion Polypeptide Components

The first component of the targeting fusion polypeptides of theinvention is a targeting ligand. A targeting ligand is a molecule, e.g.,a protein or fragment thereof that specifically binds with high affinityto a target on a pre-selected cell, such as a surface protein such as areceptor that is present to a greater degree on the pre-selected celltarget than on any other body tissue. For example, as described in U.S.Pat. Nos. 5,814,478 and 6,413,740, the MuSK receptor is highly specificto muscle. Accordingly, the cognate ligand agrin, as well as MuSKbinding portions thereof is an example of a targeting ligand useful as afirst component in the fusion polypeptides of the present invention.Another example of a targeting ligand is a group of cadherin domainsfrom a human cadherin. Accordingly, human cadherin domains from, forexample, human muscle cadherin may be used in the targeting fusionpolypeptides of the invention to target muscle cells. The targetingligand component of the fusion polypeptide of the invention may includea naturally occurring or engineered ligand, or a fragment thereof,capable of binding the pre-selected target cell.

In another embodiment of the invention, the first component targetingligand of the targeting fusion polypeptides of the invention consists ofat least three, four or five cadherin domains, or derivatives orfragments thereof, capable of binding specifically to target cells thatexpress homophilic cadherins. By the term “cadherin” is meant a moleculethat is a member of a family of calcium-dependent cell-cell adhesionmolecules consisting of several distinct proteins. Several humancadherins have been identified and characterized, including, but notlimited to, vascular endothelial cadherin (VE-cadherin; Ludwig et al.(2000) Mamm. Genome 11 (11): 1030-1033), nerve cadherin (N-cadherin;Reid et al. (1990) J. Nucleic Acids Res. 18 (19), 5896), muscle cadherin(M-cadherin; Shimoyama et al. (1998) J. Biol. Chem. 273(16):10011-10018; Shibata et al. (1997) J. Biol. Chem. 272(8):5236-5270),liver-intestine cadherin (Cadherin-17; Dantzig et al. (1994) Science264(5157):430-433), heart cadherin (Cadherin-13; Tanihara et al. (1994)Cell Adhes. Commun. 2(1):15-26), tissue-lung cadherin (Cadherin-26; Otaet al. (2004) Nat. Genet. 36(1):40-45), Cadherin-24 (Katafiasz et al.(2003) J. Biol. Chem. 278(30):27513-27519; Clark et al. (2003) GenomeRes. 13(10):2265-2270), and epithelial cadherin (E-cadherin; Cadherin-1;Bussemakers et al. (1993) Mol. Biol. Rep. 17(2):123-128).

Typically, each cadherin molecule consists of an extracellular domain,which consists of five (5) cadherin domains, as well as a transmembranedomain and intracellular domain. The extracellular cadherin domainsinteract with homophilic cadherins on adjacent cells, thus cadherins actas both ligands and receptors. Cadherins are also believed to exclude orrepel heterophilic cadherins on adjacent cells. A common feature ofhuman cadherins is the presence of 5 tandomly repeated cadherin domains.Such cadherin domains bind to homophilic cadherin domains on adjacentcells, thus causing cell-cell adhesion.

As described herein, applicants have discovered that the ability ofcadherin domains to attract homophilic cadherins can be used to createtargeting molecules that are directed to specific cell types. Further,such cell-cell adhesion can be mimicked using a minimum of three and upto five cadherin domains from any human cadherin. Accordingly,constructs are provided which utilize the specificity of cadherindomains to target the fusion polypeptides of the invention to specificcells. Thus, for example, if the desired target is muscle tissue, afusion polypeptide of the invention would comprise at least threecadherin domains from the extracellular domain of human M-cadherin (orbiologically active fragments or derivatives thereof that are capable ofbinding homophilic M-cadherin), fused to a second component that isactive on muscle cells. Alternatively, if the desired target were hearttissue, the first component would comprise at least three cadherindomains from the extracellular domain of H-cadherin, or biologicallyactive fragments or derivatives thereof that are capable of bindinghomophilic H-cadherin.

Further examples of targeting ligands also include, but are not limitedto, antibodies and portions thereof that bind a pre-selected cellssurface protein with high affinity. By “high affinity” is meant anequilibrium dissociation constant of at least 10⁻⁷ molar, as determinedby assay methods known in the art, for example, BiaCore analysis. In oneembodiment, the targeting ligand component of the targeting fusionpolypeptides of the invention may also comprise one or moreimmunoglobulin binding domains isolated from antibodies generatedagainst a selected tissue-specific surface protein or targettissue-specific receptor. The term “immunoglobulin or antibody” as usedherein refers to a mammalian, including human, polypeptide comprising aframework region from an immunoglobulin gene or fragments thereof thatspecifically binds and recognizes an antigen, which, in the case of thepresent invention, is a tissue-specific surface protein, a targettissue-specific receptor, or portion thereof. If the intended targetingfusion polypeptide will be used as a mammalian therapeutic,immunoglobulin binding regions should be derived from the correspondingmammalian immunoglobulins. If the targeting fusion polypeptide isintended for non-therapeutic use, such as for diagnostics and ELISAs,the immunoglobulin binding regions may be derived from either human ornon-human mammals, such as mice. The human immunoglobulin genes or genefragments include the kappa, lambda, alpha, gamma, delta, epsilon, andmu constant regions, as well as the myriad immunoglobulin variableregion genes. Light chains are classified as either kappa or lambda.Heavy chains are classified as gamma, mu, alpha, delta, or epsilon,which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, andIgE, respectively. Within each IgG class, there are different isotypes(e.g. IgG₁, IgG₂, etc.). Typically, the antigen-binding region of anantibody will be the most critical in determining specificity andaffinity of binding.

An exemplary immunoglobulin (antibody) structural unit of human IgG,comprises a tetramer. Each tetramer is composed of two identical pairsof polypeptide chains, each pair having one light chain (about 25 kD)and one heavy chain (about 50-70 kD). The N-terminus of each chaindefines a variable region of about 100-110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist as intact immunoglobulins, or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. For example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H) by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab)′₂ dimer intoan Fab′ monomer. The Fab′ monomer is essentially Fab with part of thehinge region. While various antibody fragments are defined in terms ofthe digestion of an intact antibody, one of skill will appreciate thatsuch fragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the terms immunoglobulin or antibody,as used herein, also includes antibody fragments either produced by themodification of whole antibodies, or those synthesized de novo usingrecombinant DNA methodologies (e.g., single chain Fv)(scFv)) or thoseidentified using phase display libraries (see, for example, McCaffertyet al. (1990) Nature 348:552-554). In addition, the fusion polypeptidesof the invention include the variable regions of the heavy (V_(H)) orthe light (V_(L)) chains of immunoglobulins, as well as tissue-specificsurface protein and target receptor-binding portions thereof. Methodsfor producing such variable regions are described in Reiter, et al.(1999) J. Mol. Biol. 290:685-698.

Methods for preparing antibodies are known to the art. See, for example,Kohler & Milstein (1975) Nature 256:495-497; Harlow & Lane (1988)Antibodies: a Laboratory Manual, Cold Spring Harbor Lab., Cold SpringHarbor, N.Y.). The genes encoding the heavy and light chains of anantibody of interest can be cloned from a cell, e.g., the genes encodinga monoclonal antibody can be cloned from a hybridoma and used to producea recombinant monoclonal antibody. Gene libraries encoding heavy andlight chains of monoclonal antibodies can also be made from hybridoma orplasma cells. Random combinations of the heavy and light chain geneproducts generate a large pool of antibodies with different antigenicspecificity. Techniques for the production of single chain antibodies orrecombinant antibodies (U.S. Pat. No. 4,946,778; U.S. Pat. No.4,816,567) can be adapted to produce antibodies used in the fusionpolypeptides and methods of the instant invention. Also, transgenicmice, or other organisms such as other mammals, may be used to expresshuman or humanized antibodies. Alternatively, phage display technologycan be used to identify antibodies, antibody fragments, such as variabledomains, and heteromeric Fab fragments that specifically bind toselected antigens.

Screening and selection of preferred immunoglobulins (antibodies) can beconducted by a variety of methods known to the art. Initial screeningfor the presence of monoclonal antibodies specific to a tissue-specificor target receptor may be conducted through the use of ELISA-basedmethods or phage display, for example. A secondary screen is preferablyconducted to identify and select a desired monoclonal antibody for usein construction of the tissue-specific fusion polypeptides of theinvention. Secondary screening may be conducted with any suitable methodknown to the art. One preferred method, termed “BiosensorModification-Assisted Profiling” (“BiaMAP”) is described in U.S. patentpublication 2004/101920, herein specifically incorporated by referencein its entirety. BiaMAP allows rapid identification of hybridoma clonesproducing monoclonal antibodies with desired characteristics. Morespecifically, monoclonal antibodies are sorted into distinctepitope-related groups based on evaluation of antibody: antigeninteractions.

Active or Therapeutic Agent

Another component of the fusion polypeptides of the invention is anactive or therapeutic agent or mutant or derivative thereof, i.e. amolecule capable of having a desired effect when delivered to thepre-selected target site, e.g., cell or tissue, Active or therapeuticagents, include, but are not limited to, small molecules, hormones,growth factors, therapeutic biologics, activating antibodies andportions thereof, and blocking antibodies and portions thereof, that arecapable of having a desirable effect upon delivery to a target cell ortissue.

In particular embodiments wherein the targeting fusion polypeptide isdirected at muscle cells or tissue, the fusion polypeptide comprises atargeting ligand, and a therapeutic agent that is active on musclecells. Such agents include, but are not limited to, insulin, IL-15,myotrophin, urocortin, urocortin II, human myostatin propeptide, IGF-1,hGH, proliferin, follistatin, FSTL1, and FLRG, or mutants, derivative,or fragments thereof having biologically activity. In addition, theactive or therapeutic agent may comprise a blocking antibody orbiologically active derivative thereof, which blocks, for example,myostatin, activin receptor, BMP receptor 1, TNF receptor and IL-1receptor. Alternatively, the active or therapeutic agent may comprise anactivating antibody that activates, for example, the IFG1 receptor,B2adrenergic receptor or the IL-15 receptor complex.

In embodiments wherein the targeting fusion polypeptide is directed atvascular endothelial cells, the fusion polypeptide comprises a targetingligand, for example, at least 3 cadherin domains of human VE-cadherinand a therapeutic agent that is active on endothelial cells, such asangiopoietin or VEGF, or activating or blocking antibodies or portionsthereof.

Optional Multimerizing Component

In specific embodiments, the fusion polypeptides of the inventioncomprise a multimerizing component. A multimerizing component includesany natural or synthetic sequence capable of interacting with anothermultimerizing component to form a higher order structure, e.g., a dimer,a trimer, etc. The multimerizing component may be selected from thegroup consisting of (i) a multimerizing component comprising a cleavableregion (C-region), (ii) a truncated multimerizing component, (iii) anamino acid sequence between 1 to about 500 amino acids in length, (iv) aleucine zipper, (v) a helix loop motif, and (vi) a coil-coil motif. Whenthe multimerizing component comprises an amino acid sequence between 1to about 500 amino acids in length, the sequence may contain one or morecysteine residues capable of forming a disulfide bond with acorresponding cysteine residue on another fusion polypeptide comprisinga multimerizing component with one or more cysteine residues. In someembodiments, the multimerizing component comprises animmunoglobulin-derived domain from, for example, human IgG, IgM or IgA,or comparable immunoglobulin domains from other animals, including, butnot limited to, mice. In specific embodiments, theimmunoglobulin-derived domain may be selected from the group consistingof the constant region of IgG, the Fc domain of IgG, an Fc-protein, theheavy chain of IgG, and the light chain of IgG. The Fc domain of IgG maybe selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as anyallotype within each isotype group.

Optional Component Spacers

The components of the targeting fusion polypeptides of the invention maybe connected directly to each other or be connected via spacers. Theterm “spacer” or “linker” means one or more molecules, e.g., nucleicacids or amino acids, or non-peptide moieties, such as polyethyleneglycol, which may be inserted between one or more component domains. Forexample, spacer sequences may be used to provide a restriction sitebetween components for ease of manipulation. A spacer may also beprovided to enhance expression of the fusion polypeptide from a hostcell, to decrease steric hindrance such that the component may assumeits optimal tertiary or quaternary structure and/or interactappropriately with its target molecule. For spacers and methods ofidentifying desirable spacers, see, for example, George et al. (2003)Protein Engineering 15:871-879, herein specifically incorporated byreference.

A spacer sequence may include one or more amino acids naturallyconnected to a receptor component, or may be an added sequence used toenhance expression of the fusion protein, provide specifically desiredsites of interest, allow component domains to form optimal tertiarystructures and/or to enhance the interaction of a component with itstarget molecule. In one embodiment, the spacer comprises one or morepeptide sequences between one or more components which is (are) between1-100 amino acids, preferably 1-25. In one specific embodiment, thespacer is a three amino acid sequence; more specifically, the threeamino acid sequence of Gly Pro Gly.

Nucleic Acid Construction and Expression

Individual components of the fusion polypeptides of the invention may beproduced from nucleic acids molecules using any suitable method known inthe art. The nucleic acids encode fusion polypeptides which comprise oneor more active or therapeutic agent(s). The nucleic acids encodetargeting fusion polypeptides which comprise (i) a component thatcomprises a ligand or derivative or fragment thereof that binds atissue-specific receptor, (ii) a component that comprises one or moreagent(s) capable of providing a therapeutic effect or activating thetarget tissue, and optionally, (iii) a multimerizing component, whereinthe multimerizing component multimerizes with a multimerizing componenton another fusion polypeptide to form a multimeric tissue-specificfusion protein of the invention. The nucleic acid molecules can beinserted into a vector that is able to express the fusion polypeptideswhen introduced into an appropriate host cell. Appropriate host cellsinclude, but are not limited to, bacterial, yeast, insect, and mammaliancells. Any of the methods known to one skilled in the art for theinsertion of DNA fragments into a vector may be used to constructexpression vectors encoding the fusion polypeptides of the inventionunder control of transcriptional/translational control signals. Thesemethods may include in vitro recombinant DNA and synthetic techniquesand in vivo recombinations (See Sambrook et al. Molecular Cloning. ALaboratory Manual, Cold Spring Harbor Laboratory; Current Protocols inMolecular Biology, Eds. Ausubel, et al., Greene Publ. Assoc.,Wiley-Interscience, NY).

Expression of the nucleic acid molecules of the invention may beregulated by a second nucleic acid sequence so that the molecule isexpressed in a host transformed with the recombinant DNA molecule. Forexample, expression of the nucleic acid molecules of the invention maybe controlled by any promoter/enhancer element known in the art.Promoters which may be used to control expression of the fusionpolypeptide molecules include, but are not limited to, the long terminalrepeat as described in Squinto et al. (1991) Cell 65:1-20; the SV40early promoter region, the CMV promoter, the M-MuLV 5′ terminal repeatthe promoter contained in the 3′ long terminal repeat of Rous sarcomavirus, the herpes thymidine kinase promoter, the regulatory sequences ofthe metallothionine gene; prokaryotic expression vectors such as theb-lactamase promoter, or the tac promoter (see also “Useful proteinsfrom recombinant bacteria” in Scientific American (1980) 242:74-94);promoter elements from yeast or fungi such as the Gal 4 promoter, theADH (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)promoter, alkaline phosphatase promoter, and tissue-specifictranscriptional control regions derived from elastase I gene, insulingene, immunoglobulin gene, mouse mammary tumor virus, albumin gene,α-fetoprotein gene, α1-antitrypsin gene, β-globin gene, myelin basicprotein gene, myosin light chain-2 gene, and gonadotropic releasinghormone gene.

In accordance with the invention, the nucleic acid constructs mayinclude components which are derived from immunoglobulins (antibodies).In general, such components will be derived from heavy (V_(H)) or light(V_(L)) chain variable regions. After identification and selection ofantibodies exhibiting the desired binding characteristics, the variableregions of the heavy chains and/or light chains of each antibody isisolated, amplified, cloned and sequenced. Modifications may be made tothe V_(H) and V_(L) nucleotide sequences, including additions ofnucleotide sequences encoding amino acids and/or carrying restrictionsites, deletions of nucleotide sequences encoding amino acids, orsubstitutions of nucleotide sequences encoding amino acids.

After identification of such ligands or portions thereof exhibitingdesired characteristics, the nucleic acids that encode such domains areused in the nucleic acid constructs. Such nucleic acids may be modified,including additions of nucleotide sequences encoding amino acids and/orcarrying restriction sites, deletions of nucleotide sequences encodingamino acids, or substitutions of nucleotide sequences encoding aminoacids.

The nucleic acid constructs of the invention are inserted into anexpression vector or viral vector by methods known to the art, whereinthe nucleic acid molecule is operatively linked to an expression controlsequence. Also provided is a host-vector system for the production of atissue-specific fusion polypeptide of the invention, which comprises theexpression vector of the invention, which has been introduced into ahost cell suitable for expression of the fusion polypeptide. Thesuitable host cell may be a bacterial cell such as E. coli, a yeastcell, such as Pichia pastoris, an insect cell, such as Spodopterafrugiperda, or a mammalian cell, such as a COS, CHO, 293, BHK or NSOcell.

The invention further encompasses methods for producing the fusionpolypeptides of the invention by growing cells transformed with anexpression vector under conditions permitting production of thetissue-specific fusion polypeptides and recovery of the fusionpolypeptides so produced. Cells may also be transduced with arecombinant virus comprising the nucleic acid construct of theinvention.

The fusion polypeptides may be purified by any technique, which allowsfor the subsequent formation of a stable polypeptide. For example, andnot by way of limitation, the fusion polypeptides may be recovered fromcells either as soluble polypeptides or as inclusion bodies, from whichthey may be extracted quantitatively by 8M guanidinium hydrochloride anddialysis. In order to further purify the fusion polypeptides,conventional ion exchange chromatography, hydrophobic interactionchromatography, reverse phase chromatography or gel filtration may beused. The fusion polypeptides may also be recovered from conditionedmedia following secretion from eukaryotic or prokaryotic cells.

In one embodiment of the invention, cells expressing a targeting fusionpolypeptide of the invention are selected having a desired highproduction rate. A variety of selection processes known to the art maybe used.

Screening and Detection Methods

The fusion polypeptides of the invention may also be used in in vitro orin vivo screening methods where it is desirable to detect and/or measuretarget protein levels or, for example, levels of receptor-bearing cells.Screening methods are well known to the art and include cell-free,cell-based, and animal assays. In vitro assays can be either solid stateor soluble. Receptor detection may be achieved in a number of ways knownto the art, including the use of a label or detectable group capable ofidentifying a tissue-specific polypeptide which is bound to a targetcell. Detectable labels are well developed in the field of immunoassaysand may generally be used in conjunction with assays using thetissue-specific fusion polypeptide of the invention.

A fusion polypeptide of the invention may also be directly or indirectlycoupled to a label or detectable group when desirable for the purpose itis being used. A wide variety of labels may be used, depending on thesensitivity required, ease of conjugation, stability requirements,available instrumentation, and disposal provisions.

Therapeutic Methods

The invention herein further provides for the development ofmuscle-targeting fusion polypeptide described herein as a therapeuticfor the treatment of patients suffering from disorders involving musclecells or tissue which express any target muscle receptor. For example, adecrease in muscle mass, or atrophy, is associated with variousphysiological and pathological states. For example, muscle atrophy canresult from denervation due to nerve trauma; degenerative, metabolic orinflammatory neuropathy, e.g. Guillian-Barre syndrome; peripheralneuropathy; or nerve damage caused by environmental toxins or drugs.Muscle atrophy may also result from denervation due to a motorneuropathy including, for example, adult motor neuron disease, such asAmyotrophic Lateral Sclerosis (ALS or Lou Gehrig's disease); infantileand juvenile spinal muscular atrophies; and autoimmune motor neuropathywith multifocal conductor block. Muscle atrophy may also result fromchronic disease resulting from, for example, paralysis due to stroke orspinal cord injury; skeletal immobilization due to trauma, such as, forexample, fracture, ligament or tendon injury, sprain or dislocation; orprolonged bed rest. Metabolic stress or nutritional insufficiency, whichmay also result in muscle atrophy, include the cachexia of cancer andother chronic illnesses including AIDS, fasting or rhabdomyolysis, andendocrine disorders such as disorders of the thyroid gland and diabetes.Muscle atrophy may also be due to a muscular dystrophy syndromes such asDuchenne, Becker, myotonic, fascioscapulohumeral, Emery-Dreifuss,oculopharyngeal, scapulohumeral, limb girdle, and congenital types, aswell as the dystrophy known as Hereditary Distal Myopathy. Muscleatrophy may also be due to a congenital myopathy, such as benigncongenital hypotonia, central core disease, nemalene myopathy, andmyotubular (centronuclear) myopathy. Muscle atrophy also occurs duringthe aging process. Muscle atrophy in various pathological states isassociated with enhanced proteolysis and decreased production of muscleproteins. Follistatin and related molecule FSTL1 and FLRG have beenshown to block myostatin-a secreted protein that inhibits muscle growth.Myostatin-inhibitors have been suggested to be useful to increase musclemass and to treat myopathy diseases (Bogdanovitch, et al. (2002) Nature420:418-421.) Accordingly, a muscle-targeting fusion protein of theinvention, wherein the active agent is folllistatin, FSTL-1 or FLRG or arelated myostatin-blocking molecule would be useful to increase musclemass or to treat myopathy diseases. In addition, human myostatinpropeptide, which also blocks myostatin, may be used to treat thesediseases.

The ability of the targeting fusion proteins of the invention to exhibita high degree of specificity for pre-selected target surface proteinsmakes them therapeutically useful for efficiently treating and/oractivating a protein(s) at a desired pre-selected site. For example, theutility of IGF-1 for the treatment of a muscle-related condition, suchas atrophy has, to date, been limited due to the presence of the IGF-1receptor in non-muscle tissue. A muscle-specific fusion polypeptide ofthe invention wherein the ligand specific for a muscle surface proteinis agrin or a MuSK-binding fragment thereof, and the active and/ortherapeutic agent is IGF-1 or fragment thereof has high degree ofspecificity for muscle tissue. IGF-I has been used to treat humanssuffering from growth hormone deficiencies, tissue wasting includingburns, skeletal trauma, infection, cancer, cystic fibrosis, Duchennemuscular dystrophy, Becker dystrophy, autosomal recessive dystrophy,polymyositis, as well as myopathies and AIDS (U.S. Pat. No. 5,622,932).

Methods of Administration

Methods known in the art for the therapeutic delivery of agents such asproteins or nucleic acids can be used for the therapeutic delivery of afusion polypeptide or a nucleic acid encoding a fusion polypeptide ofthe invention for treating and/or activating target tissue-specificsurface proteins in a subject, e.g., cellular transfection, genetherapy, direct administration with a delivery vehicle orpharmaceutically acceptable carrier, indirect delivery by providingrecombinant cells comprising a nucleic acid encoding a targeting fusionpolypeptide of the invention.

Various delivery systems are known and can be used to administer thefusion polypeptide of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987,J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part ofa retroviral or other vector, etc. Methods of introduction can beenteral or parenteral and include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, pulmonary,intranasal, intraocular, epidural, and oral routes. The compounds may beadministered by any convenient route, for example by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may beadministered together with other biologically active agents.Administration can be systemic or local. In addition, it may bedesirable to introduce the pharmaceutical compositions of the inventioninto the central nervous system by any suitable route, includingintraventricular and intrathecal injection; intraventricular injectionmay be facilitated by an intraventricular catheter, for example,attached to a reservoir, such as an Ommaya reservoir. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved, for example, and not by way oflimitation, by local infusion during surgery, topical application, e.g.,by injection, by means of a catheter, or by means of an implant, theimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, fibers, or commercial skinsubstitutes.

In another embodiment, the active agent can be delivered in a vesicle,in particular a liposome (see Langer (1990) Science 249:1527-1533). Inyet another embodiment, the active agent can be delivered in acontrolled release system. In one embodiment, a pump may be used (seeLanger (1990) supra). In another embodiment, polymeric materials can beused (see Howard et al. (1989) J. Neurosurg. 71:105). In anotherembodiment where the active agent of the invention is a nucleic acidencoding a protein, the nucleic acid can be administered in vivo topromote expression of its encoded protein, by constructing it as part ofan appropriate nucleic acid expression vector and administering it sothat it becomes intracellular, e.g., by use of a retroviral vector (see,for example, U.S. Pat. No. 4,980,286), or by direct injection, or by useof microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

Cellular Transfection and Gene Therapy

The present invention encompasses the use of nucleic acids encoding thefusion polypeptides of the invention for transfection of cells in vitroand in vivo. These nucleic acids can be inserted into any of a number ofwell-known vectors for transfection of target cells and organisms. Thenucleic acids are transfected into cells ex vivo and in vivo, throughthe interaction of the vector and the target cell. The compositions areadministered (e.g., by injection into a muscle) to a subject in anamount sufficient to elicit a therapeutic response.

In another aspect, the invention provides a method of treating a targetsite, i.e., a target cell or tissue, in a human or other animalcomprising transfecting a cell with a nucleic acid encoding atissue-specific fusion polypeptide of the invention, wherein the nucleicacid comprises an inducible promoter operably linked to the nucleic acidencoding the targeting fusion polypeptide. For gene therapy proceduresin the treatment or prevention of human disease, see for example, VanBrunt (1998) Biotechnology 6:1149-1154.

Combination Therapies

In numerous embodiments, the fusion polypeptides of the presentinvention may be administered in combination with one or more additionalcompounds or therapies. For example, multiple fusion polypeptides can beco-administered in conjunction with one or more therapeutic compounds.The combination therapy may encompass simultaneous or alternatingadministration. In addition, the combination may encompass acute orchronic administration.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositionscomprising a fusion protein of the invention and a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Suitablepharmaceutical excipients include starch, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Where necessary, thecomposition may also include a solubilizing agent and a local anestheticsuch as lidocaine to ease pain at the site of the injection. Where thecomposition is to be administered by infusion, it can be dispensed withan infusion bottle containing sterile pharmaceutical grade water orsaline. Where the composition is administered by injection, an ampouleof sterile water for injection or saline can be provided so that theingredients may be mixed prior to administration.

The active agents of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The amount of the tissue-specific fusion polypeptide of the inventionwhich will be effective in the treatment of a tissue-related conditionor disease can be determined by standard clinical techniques based onthe present description. In addition, in vitro assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness of the condition, and should bedecided according to the judgment of the practitioner and each subject'scircumstances. However, suitable dosage ranges for intravenousadministration are generally about 20-5000 micrograms of active compoundper kilogram body weight. Suitable dosage ranges for intranasaladministration are generally about 0.01 pg/kg body weight to 1 mg/kgbody weight. Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

Kits

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with at least one targeting fusion polypeptideor nucleic acid encoding a fusion polypeptide of the invention. The kitsof the invention may be used in any applicable method, including, forexample, diagnostically. Optionally associated with such container(s)can be a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice reflects (a) approval by the agency ofmanufacture, use or sale for human administration, (b) directions foruse, or both.

Transgenic Animals

The invention includes transgenic non-human animals expressing a fusionpolypeptide of the invention. A transgenic animal can be produced byintroducing nucleic acid into the male pronuclei of a fertilized oocyte,e.g., by microinjection, retroviral infection, and allowing the oocyteto develop in a pseudopregnant female foster animal. Any of theregulatory or other sequences useful in expression vectors can form partof the transgenic sequence. A tissue-specific regulatory sequence(s) canbe operably linked to the transgene to direct expression of thetransgene to particular cells. A transgenic non-human animal expressinga tissue-specific fusion polypeptide of the invention is useful in avariety of applications, including as a means of producing such fusionproteins. Further, the transgene may be placed under the control of aninducible promoter such that expression of the tissue-specific fusionpolypeptide may be controlled by, for example, administration of a smallmolecule.

Specific Embodiments

Example 1 illustrates embodiments of the fusion proteins of theinvention targeted to skeletal muscle for the treatment of a deleteriousskeletal muscle condition. More specifically described aremuscle-specific fusion proteins comprising agrin and human insulin likegrowth factor 1 (hIGF-1) and variants thereof, as well as fusionpolypeptides comprising agrin, hIGF1 and hIGF1 variants and human growthhormone (hGH) and fusion polypeptides comprising hIGF1 and variantsthereof and hGH. Example 2 describes inventions which demonstrate theuse of cadherins to produce the targeted fusion polypeptides of theinvention. Example 3 described embodiments that demonstrate the use offollistatin and related molecules as the active or therapeutic agents inthe fusion polypeptides of the invention. Example 4 describesembodiments of the fusion proteins of the invention that are active onendothelial cells. Example 5 describes myostatin-inhibiting fusionpolypeptides of the invention.

EXAMPLES

The following example is put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Muscle Targeting Fusion Polypeptides

Exemplification of the fusion polypeptides of the invention include:human IGF-1 (r3long)-linker-50 Kd COOH terminal of human agrin 0,8 (SEQID NO:1); Processed form of human IGF-1 (r3long)-linker-50 Kd COOHterminal of human agrin 0,8 (without signal sequence) (SEQ ID NO:2);hIGF-1-linker-50 Kd COOH terminal of human agrin 0,8 (without signalsequence) (SEQ ID NO:3); processed form of hIGF-1-linker-50 Kd COOHterminal of human agrin 0,8 (without signal sequence) (SEQ ID NO:4);hIGF1(r3long)-linker-50 Kd COOH terminal of human agrin 0,8 (SEQ IDNO:5); processed form of hIGF-1 (r3long)-linker-50 Kd COOH terminal ofhuman agrin 0,8 (without signal sequence) (SEQ ID NO:6); hIGF1-linker-50Kd COOH terminal of human agrin 0,0 (without signal sequence) (SEQ IDNO:7); processed form of hIGF-1-linker-50 Kd COOH terminal of humanagrin 0,0 (without signal sequence) (SEQ ID NO:8); mature human IGF1(deIGPE, deIR37)-Fc (SEQ ID NO:9); mature human IGF1(deI GPE, deIR37) (SEQID NO:10); mature human IGF1(deI GPE, deIR37) with GTG linker (SEQ IDNO:11); hGH-hIGF1-hAgrin 0,8 (SEQ ID NO:12); hGH-hIGF1-hFc (SEQ IDNO:13); hGH-hIGF1 with GTG linker (SEQ ID NO:14); hGH-hIGF2-hAgrin 0,8(SEQ ID NO:15); hGH-hIGF2-hFc (SEQ ID NO:16); hGH-hIGF2 (SEQ ID NO:17);hGH-IGF1 (SEQ ID NO:18); hGH-hIGF1 (A37) (SEQ ID NO:19); hGH-hIGF1(delete R37) (SEQ ID NO:20); hGHe-hIGF1 (mutated R3, A37) (SEQ IDNO:21); hGH-hIGF1 (mutated R3, delete R 37) (SEQ ID NO:22);hGH-hIGF1(delete R37) (SEQ ID NO:23); hGH-hIGF1 (Del GPE) (SEQ IDNO:24); hGH-hIGF1(Del GPE, del R37) (SEQ ID NO:25); hGH-hIGF1-humanagrin 0,8 (SEQ ID NO:26); hGH-hIGF1(deI GPE)-human agrin 0,8 (SEQ IDNO:27); hGH-hIGF1 (del R37)-human agrin 0,8 (SEQ ID NO:28);hGH-hIGF1(A37)-human agrin 0,8 (SEQ ID NO:29); hGH-hFc-hIGF1 (SEQ IDNO:30); hGH-hFc-hIGF1(del R37) (SEQ ID NO:31); hGH-hFc-hIGF1 (deI GPE)(SEQ ID NO:32); hGH-hFc-hIGF1 (deI GPE, del R37) (SEQ ID NO:33). Furtherembodiments include the Tercera mutant form of IGF-1 linked to humanagrin (0,8 or 0,0), and mutant forms of human IGF1 or IGF2 (such as setforth in SEQ ID NOS:10 or 11) used alone, linked to a multimerizingcomponent (such as Fc; SEQ ID NO:9) or linked to human agrin.

The fusion protein constructs or molecules (1) human growthhormone-human IGF1; (2) human growth hormone-human IGF1-human Fc; (3)human growth hormone-human IGF1-human agrin 0,8; (4) human IGF1-Fc (A3,deIR37); (5) human growth hormone and (6) human IGF1-agrin, werecompared on C2C12 myotubes for their ability to activate variousreceptors and signaling pathways. The activity of IGF-1 was measured byits ability to phosphorylate the IGF-1 receptor and Akt kinase. Theactivity of human growth hormone (hGH) was measured by its ability tophosphorylate Stat5. The activity of agrin was measured by its abilityto phosphorylate the MuSK receptor. Glass et al. (1996) 85:513-523;Beguinot et al. (1988) Biochemistry. 27(9):3222-8.

Phosphorylation assays indicated that fusion proteins having theconfiguration hGH-IGF1 which includes both hGH and hIGF1 is capable ofsimultaneously activating the IGF1 Receptor, Akt, and Stat5. Suchphosphorylation was determined by a Western blot, using phospho-specificantibodies to the various molecules, or by immune-precipitating thereceptors (such as IGFR), and Westerning with an antibody specific toanti-phospho-tyrosine. In addition, all of the above fusion polypeptideswere made using human IGF1 mutants which had the first three amino acidsdeleted (A3) and either elimination or substitution of the arginines atpositions 36 and/or 37. Such mutant IGF1 molecules demonstrated bothresistance to cleavage as well as reduced binding by IGF-1 bindingproteins (specifically IGF1 binding protein 5) without affectingsignaling ability on C2C12 myotubes.

A fusion protein which includes hGH and hIGF1 and agrin simultaneouslyactivates the IGF1 Receptor, Akt, Stat5, and the MuSK receptor, asdetermined by a Western blot, using phospho-specific antibodies to thevarious molecules, or by immune-precipitating the receptors (such asIGFR or MuSK), and Westerning with an antibody specific toanti-phospho-tyrosine. A fusion protein which includes IGF1 and agrin0,8 activates the IGF1 receptor and binds and activates the MuSKreceptor, thus providing muscle specificity. Constructs using agrin 0,0were able to bind but not activate the MuSK receptor, thus demonstratingthe use of this form of agrin for targeting muscle cells or tissue.Contacting C2C12 myotubes with the IGF1-GH fusion caused greaterhypertrophy than IGF1 alone or GH alone.

Proliferin may be substituted for hGH in any of the above constructs(Wilder, E. L. et al. (1989) Mol. Cell Biol. 9(2):430-441. Assessment ofproliferin activity may include assays for neoangiogenesis (Jackson etal. Science (1994) 266:1581-1584. The human proliferin sequence is shownin SEQ ID NO: 34.

Example 2 Muscle Targeting Fusion Polypeptides Using Cadherins

M-Cadherin containing fusion polypeptides for targeting muscle tissuewere prepared using the following amino acid sequences: IGF1-musclecadherin (SEQ ID NO:35) (containing 4 M-Cadherin domains)-IGF1; matureIGF1 (SEQ ID NO:36); full-length muscle cadherin (Cadherin 15) (SEQ IDNO:37); muscle cadherin extracellular domain containing 4 cadherindomains (SEQ ID NO:38); 4 muscle cadherin domains fused to SEAP (SEQ IDNO:39); 3 muscle cadherin domains (SEQ ID NO:40); 3 muscle cadherindomains fused to SEAP (SEQ ID NO:41); 2 muscle cadherin domains fused toSEAP (SEQ ID NO:42).

A fusion protein containing 4 N-terminal cadherin domains (SEQ ID NO:36)of human muscle cadherin (Cadherin 15) binds to skeletal musclesatellite cells. Secreted alkaline phosphatase (SeAP) was fused to theCOOH terminus of a component containing 2 (SEQ ID NO:42), 3 (SEQ IDNO:41) or 4 (SEQ ID NO:39) cadherin domains of M-cadherin. Usingantibodies to SEAP, it was determined that the fusion proteinscontaining either 3 or 4 N-terminal cadherin domains, but not the fusionprotein containing 2 cadherin domains, bound C2C12 myoblast cells, butnot fibroblasts, thus demonstrating their specificity for muscle cells.

In another embodiment, insulin-like growth factor 1 or a variant thereof(SEQ ID NOS:10 and 36) was fused to either four cadherin domains (SEQ IDNO:38) or 3 cadherin domains (SEQ ID NO:40) of M-cadherin to createmuscle-specific polypeptides. In this case, C2C12 myotubes contactedwith the fusion proteins hypertrophied, indicating that the IGF1 wasactive and that it bound to the C2C12 muscle cells. In an alternativeembodiment, two IGF-1 molecules were fused to a polypeptide comprising 4cadherin domains to create a muscle-specific fusion polypeptide (SEQ IDNO:35), which can also cause muscle hypertrophy.

Example 3 Muscle-Targeting Fusion Polypeptides Using Follistatin andRelated Molecules

The following protein sequences were also used to preparemuscle-specific fusion polypeptides that comprise themyostatin-inhibiting proteins follistatin (SEQ ID NO:43), FSTL1 (SEQ IDNO:44), and FLRG (also known as FSL3) (SEQ ID NO:45).

Follistatin, FSTLI and FLRG sequences (SEQ ID NOS: 43, 44 and 45) arefused to three or 4 N-terminal domains (SEQ ID NO: 40 or 38) ofM-cadherin. These fusion proteins all block myostatin-mediatedphosphorylation of SMAD2, indicating that they are functional. Suchfusion proteins can be used to block myostatin-mediated atrophy, ormyostatin-mediated inhibition of differentiation, with the advantage ofbeing muscle-specific.

Example 4 Vascular Endothelial Cell-Targeting Fusion Polypeptides

Fusion polypeptides comprising vascular endothelial (VE)-cadherinextracellular domains useful for targeting vascular tissue were preparedusing the following amino acid sequences, as well as the human sequenceset forth in Ludwig et al. (2000) supra. VE-cadherin Domain One (SEQ IDNO:46); VE-cadherin Domain Two (SEQ ID NO:47); VE-cadherin Domain Three(SEQ ID NO:48); VE-cadherin Domain Four (SEQ ID NO:49); and VE-cadherinDomain Five (SEQ ID NO:50).

Fusion proteins that comprise cadherin domains 1-5 (SEQ ID NOS 46-50),14 (SEQ ID NOS:46-49), and 1-3 (SEQ ID NOS:4648) of human VE cadherin(Cadherin 5), bind to vascular endothelial cells. When secreted alkalinephosphatase (SEAP) is fused to the COOH terminus of a componentcomprising four N-terminal domains of VE-cadherin (SEQ ID NOS:4649), theprotein binds endothelial cells but not fibroblasts.

In another embodiment of this example, vascular endothelial growthfactor (VEGF) is fused to amino acids 18-583 of VE-Cadherin as describedin Ludwig, D. et al. (2000) supra, which comprises SEQ ID NOS:46-49. Inthis case, endothelial cells demonstrate phosphorylation of the VEGFreceptor, and activation of Akt, indicating that the VEGF receptor isbound and activated.

Example 5 Myostatin Inhibiting Fusion Polypeptide Constructs

The following fusion polypeptides were constructed to demonstrate theuse of human myostatin propeptide in constructs which block myostatinactivity: optional 23 amino acid signal sequence, human myostatinpropeptide-human Fcl (SEQ ID NO: 51); optional 23 amino acid signalsequence, the active component is human myostatin propeptide, and thetargeting component is human agrin (SEQ ID NO:520; optional SS of 23amino acids, active agent is human myostatin propeptide, and targetingcomponent is fragment of human agrin (SEQ ID NO:53); optional SS of 23amino acids, active agent is human myostatin propeptide, andmultimerizing component is human Fc protein (SEQ ID NO:54).

C2C12 myoblasts are grown to confluence and differentiated intomyotubes. Differentiation is accomplished by switching todifferentiation media—2% Horse Serum, in DMEM. Forty eight (48) toninety six (96) hours post-differentiation, the myotubes are starved forfour (4) hours, in serum-free media. For this experiment, plates ofmyotubes are treated in groups of two, for fifteen minutes, as follows:(a) untreated, (b) 10 ng/ml active myostatin, (c) a myostatin inhibitorof the instant invention (for example, the pre-pro domain of myostatinfused to an Fc, or the pre-pro domain of myostatin fused to agrin), atconcentrations which could range from 10 ng/ml to 10 ug/ml (d) activemyostatin plus a myostatin inhibitor of the instant invention. Aftertreatment, the myotubes are lysed, for example, in NP40 lysis buffercontaining phosphatase and protease inhibitors. The resulting lysatesare then processed for Western blot analysis with an antibody specificfor phosphorylated Smad2, and, as a control for total protein levels, asecond Western was performed using an antibody which recognizesnonphosphorylated Smad2. The assay demonstrates that myostatin (GDF8)induces SMAD 2 phosphorylation and that the myostatin inhibitorsdescribed above block myostatin-mediated phosphorylation of Smad2.

1. A targeting fusion polypeptide, comprising: (i) a first componentcomprising a targeting ligand, or derivative or fragment thereof,capable of binding specifically to a pre-selected cell surface protein;(ii) a second component comprising at least one active or therapeuticagent; and optionally, (iii) a multimerizing component capable offorming a multimer with another targeting fusion polypeptide; and/or(iv) a signal sequence.
 2. The targeting fusion polypeptide of claim 1,wherein (i) is three N-terminal cadherin domains of a human cadherin. 3.The targeting fusion polypeptide of claim 2, wherein the human cadherinis selected from the group consisting of muscle cadherin (M-cadherin),nerve cadherin (N-cadherin), vascular endothelial cadherin(VE-cadherin), cadherin 17 (liver-intestine cadherin), cadherin 13(heart cadherin), cadherin 26, cadherin 24 and epithelial cadherin(E-cadherin or cadherin-1).
 4. The targeting fusion polypeptide of claim3, wherein (i) is 3 cadherin domains of human VE-cadherin.
 5. Thetargeting fusion polypeptide of claim 4, wherein (ii) is selected fromthe group consisting of vascular endothelial growth factor (VEGF),angiopoietin-1, or angiopoietin-2, or mutants, derivatives or fragmentsthereof having biological activity.
 6. A muscle-targeting fusionpolypeptide, comprising the targeting fusion polypeptide of claim 1,wherein wherein the pre-selected cell surface protein is a muscle cellsurface protein.
 7. The muscle-targeting fusion polypeptide of claim 7,wherein the muscle surface protein is a receptor.
 8. Themuscle-targeting fusion polypeptide of claim 7, wherein the receptor isMuSK and the targeting ligand is one of agrin, a fragment of agrincapable of binding the MuSK receptor, an anti-MuSK antibody, or ananti-Musk antibody fragment or derivative thereof having biologicalactivity.
 9. The muscle-targeting fusion polypeptide of claim 8, wherein(ii) is a ligand for a cell surface receptor, and is capable of bindingand activating the cell surface receptor.
 10. The targeting fusionpolypeptide of claim 1, wherein the multimerizing component is selectedfrom the group consisting of (i) a multimerizing component comprising acleavable region (C-region), (ii) a truncated multimerizing component,(iii) an amino acid sequence between 1 to about 500 amino acids inlength, optionally comprising at least one cysteine residue, (iv) aleucine zipper, (v) a helix loop motif, (vi) a coil-coil motif, and(vii) an Fc-protein.
 11. A multimeric fusion polypeptide comprising twoor more of the targeting fusion polypeptides of claim
 10. 12. Apharmaceutical composition comprising the fusion polypeptide of claim 6and a pharmaceutically acceptable carrier.
 13. A method of treating,ameliorating, or inhibiting a muscle-related condition or disease in asubject in need thereof, comprising administering the pharmaceuticalcomposition of claim
 12. 14. The method of claim 14, wherein thecondition or disease is muscle atrophy or muscle dystrophy.
 15. Anisolated nucleic acid molecule encoding the fusion polypeptide of claim6.
 16. A combined therapeutic fusion polypeptide, comprising (i) a firstactive or therapeutic agent, and (ii) a second active or therapeuticagent; optionally further comprising (iii) a multimerizing componentcapable of forming a multimer with another therapeutic fusionpolypeptide, and/or (iv) a signal sequence.
 17. The combined fusionpolypeptide of claim 16, wherein the first or second active ortherapeutic agent is an antibody.
 18. The combined fusion polypeptide ofclaim 17, wherein the antibody is a blocking antibody specific for anantigen selected from the group consisting of myostatin, activinreceptor, bone morphogenic protein (BMP) receptor1, tumor necrosisfactor (TNF) receptor and interleukin-1 (IL-1) receptor.
 19. Thecombined fusion polypeptide of claim 17, wherein the antibody is anactivating antibody specific for an antigen selected from the groupconsisting of insulin-like growth factor-1 (IGF1) receptor, B2adrenergicreceptor and the IL-15 receptor complex.
 20. The combined fusionpolypeptide of claim 16, wherein the first and/or second active ortherapeutic agent is a ligand for a cell surface receptor, and iscapable of binding and activating the cell surface receptor.
 21. Thecombined fusion polypeptide of claim 20, wherein the first and/or secondactive or therapeutic agent is selected from the group consisting ofIL-15, myotrophin, urocortin, urocortin II, a natural or mutant humanIGF1 or IGF2, insulin, human myostatin propeptide, human growth hormone(hGH), proliferin, follistatin, FSTL1, and FLRG, or a mutant,derivative, or fragment thereof having biologically activity.
 22. Thecombined fusion polypeptide of claim 21, wherein the mutant human IGF1comprises (i) a modification selected from the group consisting ofdeIGPE (1-3), E3R or E3A, and (ii) a modification selected from thegroup consisting of R36A, R37A, del36 or del37.
 23. The combined fusionpolypeptide of claim 22, wherein the first active or therapeutic agentis hGH.
 24. The combined fusion polypeptide of claim 16, wherein themultimerizing component is selected from the group consisting of (i) amultimerizing component comprising a cleavable region (C-region), (ii) atruncated multimerizing component, (iii) an amino acid sequence between1 to about 500 amino acids in length, optionally comprising at least onecysteine residue, (iv) a leucine zipper, (v) a helix loop motif, (vi) acoil-coil motif, and (vii) an Fc-protein.
 25. A multimeric fusionpolypeptide comprising two or more of the targeting fusion polypeptidesof claim
 24. 26. An insulin-like growth factor-1 (IGF1)-related fusionpolypeptide, comprising (i) human IGF1 comprising: (a) a modificationselected from the group consisting of delGPE (1-3), E3R or E3A; and (b)a modification selected from the group consisting of R36A, R37A, del36or del37; (ii) a multimerizing component capable of forming a multimerwith another IGF-related polypeptide, wherein the IGF1-related fusionpolypeptide optionally comprises (iii) a signal sequence.
 27. Anisolated nucleic acid molecule encoding the fusion polypeptide of claim26.
 28. A myostatin inhibiting fusion polypeptide comprising (P)_(x)−M,wherein P is human myostatin propeptide, or a fragment or variantthereof capable of binding and inhibiting the biological activity ofmyostatin, and M is a mutimerizing component, wherein X is a numberbetween 1-10.
 29. An isolated nucleic acid molecule encoding the fusionpolypeptide of claim 28.